We have previously shown that in the particulate fraction of rat renal cortex there is a protein activator of cyclic nucleotide phosphodiesterase, which can be released by hypotonic lysis. The kidney phosphodiesterase activator has now been partially purified and characterized. It is a thiol protease of lysosomal origin. With regard to its lysosomal location, thiol requirement, molecular weight (approximately 30,000) and other properties kidney phosphodiesterase activator resembles cathepsin B. The purified activator is inhibited by the bacterial peptides leupeptin and antipain, which also block the activation of phosphodiesterase completely in hypotonic homogenates of rat kidney, and partially in frozen-thawed homogenates of rat liver. Whether or not this activator participates in the control of cyclic nucleotide phosphodiesterase in situ remains to be established.